Poxviruses provide a unique system for studying the replication of DNA. Required enzymes and factors are encoded within the viral genome and DNA synthesis and processing occurs within the cytoplasmic compartment of the cell. Therefore, it has been possible to apply genetic and biochemical approaches to the study of DNA replication. Our effort has been directed towards ascertaining the structure and mode of replication of the poxvirus genome with particular emphasis placed on understanding the processing of the replicative intermediates. The replication of vaccinia virus proceeds through concatemeric highly branched intermediates that are resolved into unit length DNA molecules. Mutational analysis has demonstrated that a cis acting DNA sequence highly conserved among poxviruses as well as the palindromic structure of the concatemer is essential for resolution of the telomere and that resolution occurs by a process involving conservative strand exchange. A model for resolution involving site-specific recombination and oriented branch migration is consistent with this data. A separate sequence independent mechanism is responsible for the resolution of the numerous branch points present in the replicative intermediates. Our present efforts are directed towards determining the trans acting protein components that participate in telomeric as well as branch resolution. An open reading frame has been identified in vaccinia that encodes for a gene with homology to site-specific recombination proteins. This gene is essential for viral growth, synthesized in small amounts after DNA replication, and myristilated. We are trying to determine the function of this gene during vaccinia virus replication. An activity has been detected in virion extracts that cleaves and cross-links DNA. This activity is dependent on DNA structure, independent of ATP and metal ion concentrations and is similar to activities reported for enzymes that process branched DNA structures. The activity co-chromatographs with a specific viral protein. We are confirming the tentative identification of the viral protein responsible for this activity and investigating its role in vaccinia virus DNA processing.